The present invention relates to the crystal structure of the cfms kinase domain, specifically the cFMS kinase domain in liganded form, methods of using the same in the discovery of cFMS inhibitors and in the treatment of diseases mediated by inappropriate cfms activity, as well as diamino-pyrimidine cFMS inhibitors.
Colony stimulating factor-1 receptor (CSF-1R or CFMS), encoded by the proto oncogene c-fms (Sherr 1985; Woolford 1985), is a cell surface receptor for the macrophage colony-stimulating factor (M-CSF or CSF-1) and a member of the receptor tyrosine kinase (RTK) family of growth factor receptors. CSF-1 (or M-CSF) is a homodimeric growth factor involved in the proliferation, differentiation, and activation of monocytes or macrophages, as well as a proinflammatory cytokine (Tushinski 1982; Metcalf 1986; Becker 1987; Elliott 1989; Young 1990).
cFMS is a hematopoietic growth factor receptor, whose expression is lineage-specific and primarily confined to monocytes, macrophages and their bone marrow progenitors. cFMS is the cellular counterpart of the v-fms oncogene encoded by the genomes of both Susan McDonough and Hardy-Zuckerman five strains of feline sarcoma virus (Coussens 1986; Sherr 1988). The receptor is comprised of an extracellular ligand-binding domain joined through a single membrane-spanning helix to an intracellular protein tyrosine kinase (PTK) domain. It is closely related structurally to the receptors for the platelet-derived growth factor (PDGF), the stem cell factor receptor (the c-kit proto-oncogene product), and flt3/flt2. The extracellular ligand binding domain of these receptors is composed of five immunoglobulin-like loops (Wang 1993), and the PTK domains contain kinase insert (KI) sequences of varying lengths (Sherr, 1991). Binding of CSF-1 to the receptor extracellular ligand-binding domain causes a conformational change and induces a noncovalent dimerization of cFMS, autophosphorylation and activation of the PTK domain, and trans-phosphorylation of specific tyrosine residues in the cytoplasmic domain. These phosphorylated tyrosine residues serve as binding sites for src-homology 2 (SH2) domains contained within cytoplasmic signaling proteins (Sengupta 1988; Reedijk 1992), thereby activating signaling cascades. Both the PI3K-dependent and Ras/mitogen-activated protein kinase-dependent pathways are activated in response to CSF-1 binding to cFMS (Yeung 1998; Kelley, 1999; Kanagasundaram 1999). Following activation of cFMS with CSF-1, the receptor is rapidly internalized via clathrin-coated pits and vesicles and targeted to the lysosome for degradation.
cFMS receptor expression in macrophage populations corresponds to its stage of differentiation and tissue localization. CSF-1/cFMS interaction and signaling is required for the recruitment, development, and maintenance of a subset of macrophages, such as marrow and blood monocytes. Thus, an absence of cFMS is not life-threatening. Deletion of CSF-1, as occurs in the mutant mouse strain op/op and in the mutant rat strain th1/th1, provides an insight into the biology of cFMS/CSF-1 signaling. The op/op mutation renders an osteopetrotic phenotype with severely deficient macrophage populations in the joints, osteoclasts, peritoneal cavity phagocytes, splenic marginal zone metallophils, and lymph node subcapsular sinus macrophages. Other populations reach substantial levels, including bone marrow, phagocytes in the thymic cortex, splenic red pulp, lymph node medulla, intestinal lamina propria, liver (Kupffer cells), lung (alveolar macrophages), and brain (microglia) (Yoshida, H 1990; Wiktor-Jedrzejczak, W. 1991).
Connective tissue macrophages are involved in a number of chronic disease states, such as osteoarthritis, rheumatoid arthritis, osteoporosis, cardiovascular/vessel-wall disease, chronic graft rejection, Alzheimer's, and Lupus-nephritis (Yang 2001; Bischof, 2000; Boyce 1999; Cenci 2000; Campbell 2000; Murphy 2000). These macrophages directly or indirectly influence the production of disease modifiers (MMPs, cathepsins, chemokines, growth and differentiation factors) within the microenvironment (Valledor 2000).
Endogenous CSF-1 critically regulates HIV-1 replication in human monocyte-derived macrophages (MDM). The HIV-1 infected MDM cells produce high levels of CSF-1 by a mechanism that requires active virus replication. This aids the survival of infected macrophages and enhances the spread of infection by increasing macrophage susceptibility to the HIV virus. This suggests that CSF-1 might be a therapeutic target to block HIV-1 replication in human macrophages (Kalter 1991; Bergamini 1994; Gallo 1994; Kutza 2000).
Cancer studies have revealed elevated levels of circulating CSF-1 in patients with acute myeloid leukemia (AML) (Haran-Ghera 1997). It has also been reported that CSF-1 gene transduction into human lung carcinoma cell lines resulted in inhibition of metastic disease to the liver and lymph nodes, but not to the kidney. This suggests that the heterogeneity of organ microenvironments influences the spread of lung carcinoma (Yano 1997). Other evidence suggests that c-Fms activation by CSF-1 induces invasive disease by a urokinase-dependent pathway in breast carcinoma and neoplasms of the female reproductive tract (Kacinski 1997).
Inhibition of the c-fms receptor kinase represents a novel approach in the treatment of chronic disease by modulating proliferation, activation, differentiation, and migration of specific subpopulations of macrophages. It is envisioned that such a treatment would lead to a disease modifying effect—thereby alleviating signs and symptoms. Immunocompetence may be less compromised by this approach than by a more global inflammatory mediator depletion or immune suppressive approaches. Determination of a crystal structure of the cFMS kinase domain would provide a useful tool for indentifying ihibitors of cFMS.
The present inventors have determined the crystal structure of the cFMS kinase domain (cFMSK) alone and complexed with a cFMS inhibitor to 2.7 and 1.8 Å resolution, respectively. The crystal structure contains the non-phosphorylated, catalytic core as well as an N-terminal, juxtamembrane region (NT region). Such a crystal structure is useful in discovering compounds suitable for inhibiting cFMS and for treating diseases characterized by aberrant cfms activity. Also included in the present invention are diamino-pyrimidine cfms inhibitors.